The overall goal of these studies is to understand how endothelial nitric oxide synthase (eNOS) signaling is regulated in the pulmonary system with emphasis on its role in the perinatal period. Initially considered to be a constitutively expressed enzyme, it is now apparent that eNOS is dynamically regulated at the transcriptional, post-transcriptional, post-translational, and developmental levels. During the previous funding period we explored the regulation of eNOS in the pulmonary system by fluid shear stress in the perinatal period. We identified a novel shear stress response pathway regulating eNOS expression via PKC and JNK/c-Jun in pulmonary arterial endothelial cells (PAECs) isolated from late gestation fetal lambs. Further, we demonstrated that PAECs isolated from adult animals had less basal eNOS expression than those from late gestation fetal animals. We have also shown that basal NO generation in fetal PAECs is regulated by estrogen. In addition, we found that PAECs isolated from juvenile animals, although expressing eNOS at levels comparable to those derived from fetal PAECs, generate less NO and more reactive oxygen species (ROS) when stimulated. This appears to be due to a decrease in cellular tetrahydrobiopterin (BH4). Our new data indicate this decrease in BH4 levels appears to be mediated by a loss of estrogen responsiveness leading to a decrease in GTP cyclohydrolase I expression (the rate limiting enzyme in BH4 biosynthesis). Thus, the overall hypothesis we will test in this application is that estrogen plays an important role in the mechanisms by which eNOS activity and gene expression are regulated in the pulmonary system. We plan to characterize how "coupling" of eNOS is modulated to produce either NO or ROS and determine if ROS have functional effects on soluble guanylate cyclase activity. We will also explore the mechanisms by which estrogen regulates eNOS activity and gene expression. Finally, we will determine whether there is a coordinated regulation of eNOS and GTP cyclohydrolase I mediated by estrogen induced NO generation in fetal PAECs. It is anticipated that the successful completion of these studies will increase our understanding of the role played by estrogen in regulating eNOS activity and expression in the pulmonary system in the perinatal period. [unreadable] [unreadable]